H2O2 activates G protein, α 12 to disrupt the junctional complex and enhance ischemia reperfusion injury

The epithelial cell tight junction separates apical and basolateral domains and is essential for barrier function. Disruption of the tight junction is a hallmark of epithelial cell damage and can lead to end organ damage including renal failure. Herein, we identify G alpha 12 activation by H2O2 leading to tight junction disruption and demonstrate a critical role for G alpha 12 activation during bilateral renal ischemia/reperfusion injury. Madin-Darby canine kidney (MDCK) cells with inducible G alpha 12 (G alpha 12-MDCK) and silenced G alpha 12 (shG alpha 12-MDCK) were subjected to ATP depletion/repletion and H2O2/catalase as models of tight junction disruption and recovery by monitoring transepithelial resistance. In ATP depleted cells, barrier disruption and recovery was not affected by G alpha 12, but reassembly was accelerated by G alpha 12 depletion. In contrast, silencing of G alpha 12 completely protected cells from H2O2-stimulated barrier disruption, a response that rapidly occurred in control cells. H2O2 activated Src and Rho, and Src inhibition (by PP2), but not Rho (by Y27632), protected cells from H2O2-mediated barrier disruption. Immunofluorescent and biochemical analysis showed that H2O2 led to increased tyrosine phosphorylation of numerous proteins and altered membrane localization of tight junction proteins through G alpha 12/Src signaling pathway. G alpha 12 and Src were activated in vivo during ischemia/reperfusion injury, and transgenic mice with renal tubular QL alpha 12 (activated mutant) expression were delayed in recovery and showed more extensive injury. Conversely, G alpha 12 knockout mice were nearly completely protected from ischemia/ reperfusion injury. Taken together, these studies reveal that ROS stimulates G alpha 12 to activate injury pathways and identifies a therapeutic target for ameliorating ROS mediated injury.